The cells of tissues are generally in contact with a network of large extracellular macromolecules that occupies the spaces in a tissue between the component cells and also occupies the space between adjacent tissues. This extracellular matrix functions as a scaffolding on which the cells and tissue are supported and is involved actively in regulating interaction of the cells that contact it. The principal macromolecules of the extracellular matrix include the collagens (the most abundant proteins in the body) and glycosaminoglycans (complex polysaccharides which are usually bonded also to protein and then termed proteoglycans). The macromolecules that comprise the extracellular matrix are produced typically by the cells in contact therewith, for example, epithelial cells in contact with a basement membrane and fibroblasts embedded in connective tissue.
The glycosaminoglycan (proteoglycan) molecules form a highly hydrated matrix (a gel) in which elastic or fibrous proteins (such as collagen fibers) are embedded. The aqueous nature of the gel permits diffusion of metabolically required substances between the cells of a tissue and between tissues. Additional proteins that may be found in extracellular matrix include elastin, fibronectin and laminin.
The term “connective tissue” refers to extracellular matrix plus specialised cells such as, for example, fibroblasts, chondrocytes, osteoblasts, macrophages and mast cells found therein. The term “interstitial tissue” is best reserved for an extracellular matrix that stabilizes a tissue internally, filling the gaps between the cells thereof. There are also specialized forms of extracellular matrix (connective tissue) that have additional functional roles—cornea, cartilage and tendon, and when calcified, the bones and teeth.
A structural form of extracellular matrix is the basal lamina (basement membrane). Basal laminae are thin zones of extracellular matrix that are found under epithelium or surrounding, for example, muscle cells or the cells that electrically insulate nerve fibres. Generally speaking, basal laminae separate cell layers from underlying zones of connective tissue or serve as a boundary between two cell layers wherein a basal lamina can serve as a pathway for invading cells associated with pathologic processes, or for structural organisation associated with tissue repair (i.e. as a blueprint from which to regenerate original tissue architecture and morphology).
The regulated turnover of extracellular matrix macromolecules is critical to a variety of important biological processes. Localised degradation of matrix components is required when cells migrate through a basal lamina, as when white blood cells migrate across the vascular basal lamina into tissues in response to infection or injury, or when cancer cells migrate from their site of origin to distant organs via the bloodstream or lymphatic vessels, during metastasis. In normal tissues, the activity of extracellular proteases is tightly regulated and the breakdown/production of connective tissue is in dynamic equilibrium, such that there is a slow and continual turnover due to degradation and resynthesis in the extracellular matrix of adult animals.
In each of these cases, matrix components are degraded by extracellular proteolytic enzymes that are secreted locally by cells. These proteases belong to one of four general classes: many are metalloproteinases, which depend on bound Ca.sup.2+ or Zn.sup.2+ for activity, while the others are serine, aspartic and cysteine proteases, which have a highly reactive serine, aspartate or cysteine residue in their respective active site (Vincenti et al., (1994) Arthritis and Rheumatism, 37: 1115-1126). Together, metalloproteinases, serine, aspartate and cysteine proteases cooperate to degrade matrix proteins such as collagen, laminin, and fibronectin.
Several mechanisms operate to ensure that the degradation of matrix components is tightly controlled. First, many proteases are secreted as inactive precursors that can be activated locally. Second, the action of proteases is confined to specific areas by various secreted protease inhibitors, such as the tissue inhibitors of metalloproteases and the serine protease inhibitors known as serpins. These inhibitors are specific for particular proteases and bind tightly to the activated enzyme to block its activity. Third, many cells have receptors on their surface that bind proteases, thereby confining the enzyme to where it is needed.
Many pathogenic bacteria produce extracellular metalloproteases, of which many are zinc containing proteases that can be classified into two families, the thermolysin (neutral) proteases and the serralysin (alkaline) proteases.
A number of patents and publications report the inhibition of one or more extracellular proteases by compounds extracted from plants. For example, Sun et al., (1996) Phytotherapy Res., 10: 194-197, reports the inhibition in vitro of stromelysin (MMP-3) and collagenase by betulinic acid extracted from Doliocarpus verruculosis. Sazuka et al, (1997).
Biosci. Biotechnol. Biochem., 61: 1504-1506, reports the inhibition of gelatinases (MMP-2 and MMP-9) and metastasis by compounds isolated from green and black teas. Kumagai et al, JP 08104628 A2, Apr. 1, 1996 (CA 125: 67741) reports the use of flavones and anthocyanines isolated from Scutellaris baicanlensis roots to inhibit collagenase. Gervasi et al., (1996) Biochem. Biophys. Res. Comm., 228: 530-538, reports the regulation of MMP-2 by some plant lectins and other saccharides. Dubois et al., (1998) FEBS Lett., 427: 275-278, reports the increased secretion of deletorious gelatinase-B (MMP-9) by some plant lectins. Nagase et al., (1998) Planta Med., 64: 216-219, reports the weak inhibition of collagenase (MMPs) by delphinidin, a flavonoid isolated from Solanum melongena. 
Other reports discuss the use of extracts to inhibit extracellular proteases. For example, Asano et al., (1998) Immunopharmacology, 39: 117-126, reports the inhibition of TNF-a production using Tripteggium wilfordii Hook F. extracts. Maheu et al., (1998) Arthritis Rheumatol., 41: 81-91, reports the use of avocado/soy bean non-saponifiable extracts in the treatment of arthritis. Makimura et al., (1993). J. Periodontol., 64: 630-636, also reports the use of green tea extracts to inhibit collagenases in vitro. Obayashi et al., (1998) Nippon Keshonin Gijutsusha Kaishi, 32: 272-279 (CA 130: 92196) reports the inhibition of collagenase-I (MMP-1) from human fibroblast and neutrophil elastase by plant extract from Eucalyptus and Elder.
When a plant is stressed, several biochemical processes are activated and many new chemicals, in addition to those constitutively expressed, are synthesised as a response. These chemicals include enzymes, enzyme inhibitors (especially protease inhibitors), lectins, alkaloids, terpenes, oligosaccharides, and antibiotics. The biosynthesis of these defense chemicals and secondary metabolites is not yet fully understood. The most studied system is the production of protease inhibitors following pest attack or mechanical wounding. On the other hand, several inducible chemicals are the products of complex biochemical pathways which require several biosynthetic enzymes to be activated.
It has been shown that many chemicals can be used to “stress” plants and to artificially stimulate biosynthesis of several new and constitutive defense chemicals. Also, different types of stress can activate distinct metabolic defense pathways, thereby leading to production of a variety of chemicals. Although the various biosynthetic defense pathways share some similarities, these pathways are characteristic of specific plant species. Therefore, treating many plants with many types of stress can lead to a vast number of collections of diverse chemicals from plant origin.
In addition to pests, fungi, and other pathogenic attacks, stressors include drought, heat, water and mechanical wounding. Furthermore, many chemicals can act as stressors that activate gene expression; these include: hydrogen peroxide, ozone, sodium chloride, jasmonic acid and derivatives, .alpha.-linoleic acid, ‘.gamma.-linoleic acid, salicylic acid, abscesic acid, volicitin, small oligopeptides, among others.
The use of abiotic stressors on plants has been the focus of intense studies in plant science. Artificial stresses have been used to stimulate the production of natural plant protease inhibitors for insect digestive proteases, in order to enhance crop protection against certain pests and herbivores. They have proven useful in combination with plants genetically modified to express other protease inhibitor genes. Finally, in the area of molecular farming, stresses have been used to stimulate gene expression in plants genetically modified to include an inducible coding sequence for a protein of nutraceutical and/or medicinal interest (Ryan and Farmer, U.S. Pat. No. 5,935,809).
Likewise, the use of gene activators or elicitors have been described to enhance the production of volatile chemicals in plant cell cultures. These elicitors have been demonstrated to induce the activity of several enzymes such as for example phenylalanine ammonia lyase, therefore leading to an increase in the production of plant volatile components.
No one has used stress to improve or modify plants human protease inhibitor content.
Table 1 reports the inhibition of human MMP-1 by aqueous (A), ethanolic (R) and organic (S) 25 extracts for exemplary stressed and non-stressed plant sources.
Table 2 reports the inhibition of human MMP-2 by aqueous (A), ethanolic (R) and organic (S) extracts for exemplary stressed and non-stressed plant sources.
Table 4 reports the inhibition of human MMP-9 by aqueous (A), ethanolic (R) and organic (S) extracts for exemplary stressed and non-stressed plant sources.
Table 5 reports the inhibition of human Cathepsin B by aqueous (A), ethanolic (R) and organic (S) extracts for exemplary stressed and non-stressed plant sources.
Table 6 reports the inhibition of human Cathepsin D by aqueous (A), ethanolic (R) and organic (S) extracts for exemplary stressed and non-stressed plant sources.
Table 7 reports the inhibition of human Cathepsin G by aqueous (A), ethanolic (R) and organic (S) extracts for exemplary stressed and non-stressed plant sources.
Table 8 reports the inhibition of human Cathepsin L by aqueous (A), ethanolic (R) and organic (S) extracts for exemplary stressed and non-stressed plant sources.
Table 9 reports the inhibition of human Cathepsin K by aqueous (A), ethanolic (R) and organic (S) extracts for exemplary stressed and non-stressed plant sources.
Table 10 reports the inhibition of FILE by aqueous (A), ethanolic (R) and organic (S) extracts for 20 exemplary stressed and non-stressed plant sources.
Table 11 reports the inhibition of bacteria Clostripain by aqueous (A), ethanolic (R) and organic (S) extracts for exemplary stressed and non-stressed plant sources.
Table 12 reports the inhibition of bacteria subtilisin by aqueous (A), ethanolic (R) and organic (S) extracts for exemplary stressed and non-stressed plant sources.